Chemosphere 76 (2009) 711715

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Chemosphere
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Technical Note

Lead chelation to immobilised Symphytum ofcinale L. (comfrey) root tannins

Lily Chin *, David W.M. Leung *, H. Harry Taylor
School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand

a r t i c l e

i n f o

a b s t r a c t
Reported correlations between tannin level and metal accumulation within plant tissues suggest that metal-chelating tannins may help plants to tolerate toxic levels of heavy metal contaminants. This paper supports such correlations using a new method that demonstrated the ability of plant tannins to chelate heavy metals, and showed that the relative levels of tannins in tissues were quantitatively related to lead chelation in vitro. Using this in vitro metal chelation method, we showed that immobilised tannins prepared from lateral roots of Symphytum ofcinale L., that contained high tannin levels, chelated 3.5 times more lead than those from main roots with lower tannin levels. This trend was conrmed using increasing concentrations of tannins from a single root type, and using puried tannins (tannic acid) from Chinese gallnuts. This study presents a new, simple, and reliable method that demonstrates direct leadtannin chelation. In relation to phytoremediation, it also suggests that plant roots with more built-in tannins may advantageously accumulate more lead. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 29 November 2008 Received in revised form 26 April 2009 Accepted 27 April 2009 Available online 27 May 2009 Keywords: Lead Tolerance Phytoremediation Polyphenols PVPP

1. Introduction Phytoremediation is a technology that uses plants to remove or render harmless pollutants in the environment (Garbisu and Alkorta, 2001). Lead is often considered as the target pollutant of remediation studies due to its widespread distribution, persistence, and toxicity to human health (Chen et al., 1997). Moreover, Pb is ranked the number one heavy metal pollutant (and number two of all hazardous substances) by the Agency for Toxic Substances and Disease Registry (ATSDR, 2005). However, the maximum heavy metal remediation potential of plants is yet to be realised. To address this need, one avenue is to gain a better understanding of heavy metal tolerance in plants. Some notable examples of heavy metal tolerance have been linked with enhanced metal accumulation, although this is not always observed (Yang et al., 2004; Cherian and Oliveira, 2005). There are two principal types of heavy metal tolerance mechanisms. The rst mechanism serves to prevent heavy metal stress by controlling the concentration of free metal-ions in the cell (or tissue) as a result of their isolation in vacuoles and vesicles, or by ligand-based chelation (Wierzbicka, 1995; Hall, 2002). The second mechanism involves detoxifying and repairing the effects of reactive oxygen species created during heavy metal stress, using antioxidants and enzymes such as superoxide dismutase (Schtzendbel et al., 2001).

* Corresponding authors. Tel.: +64 3 364 2650; fax: +64 3 364 2590. E-mail addresses: lchin.email@gmail.com (L. Chin), david.leung@canterbury.ac.nz (D.W.M. Leung). 0045-6535/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.04.059

This study focused on ligand-based chelation of potentially harmful free metals in the apoplast or cytosol (Yang et al., 2004). Organic ligands such as phytochelatins, organic acids, amino acids, cell wall proteins (all reviewed by Hall, 2002), cell wall pectins (Thurman, 1981), and polyphenols (Lavid et al., 2001), have been the focus of most heavy metal chelation studies involving plants or micro-organisms. In the present study we are interested the plant polyphenols, particularly high molecular weight polyphenols called tannins, as organic ligands for metal chelation. Polyphenols consist of 6-C aromatic ring(s) with one or more hydroxyl groups bound to it (Waterman and Mole, 1994). These hydroxyl groups form stable complexes with metal ions when two are positioned side by side on the aromatic ring. This is a well-known property which has led to the development of copper-based wood preservatives (Laks and Mckaig, 1988) and anti-microbial agents (Scalbert et al., 1999), and to studies on the effects of polyphenols in human and cattle nutrition (Bravo, 1998). According to dietary studies by Jackson et al. (1996), low and high tannin levels in plant tissues are considered to be approximately 2% and 6% (dw), respectively. Metal accumulation in plants is known to be positively correlated with the levels of tannins in various plant tissues (Lavid et al., 2001; Kamachi et al., 2005) and with high molecular weight polyphenols exuded by plant roots (Jung et al., 2003). Moreover, total polyphenol levels of plant tissues have been positively correlated with metal accumulation (Tripathi and Tripathi, 1999; Ruso et al., 2001). Because of the great diversity in tannin structures (Kraus et al., 2003), the chelating properties of tannin in plant tissues cannot be conclusively inferred from tannin or polyphenol levels alone.

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Direct chelation experiments are necessary and would shed new insights into this tolerance mechanism. Dialysis (Lavid et al., 2001) and electron energy loss spectroscopy (Neumann et al., 1995) are two of the few reported methods that have shown metal chelation in vivo or to crude extracts of polyphenols. Dialysis is the simplest of these. In this method, dialysis tubes containing polyphenol extracts are placed in metal solutions; it is expected that higher tannin concentrations in the dialysis tubes would trap greater quantities of Pb by chelation inside the tubes. However, dialysis tubes would trap all high molecular weight plant compounds and would not distinguish between polyphenols and other metal-chelating compounds, such as proteins and polysaccharides in the crude extract. While purication of the polyphenol extracts and the use of immobilised tannins based on complicated established methods (Nakajima and Sakaguchi, 2000; Liao et al., 2004) may solve this issue, this approach would be time-consuming and costly. The purpose of the present research was to devise a simple and reliable in vitro metal chelation method to evaluate the chelation properties of tannin extracts and, in particular, to use this new method to test the correlation between the level of built-in tannins in tissues and metal accumulation. Central to this method was the use of insoluble polyvinyl polypyrrolidone (PVPP). Unlike dialysis, PVPP can isolate and simply immobilise tannins. At a laboratory scale, PVPP is used to isolate tannins in polyphenols assays (FAO/IAEA Manual, 2002) and to commonly remove enzyme-inhibiting polyphenols during enzyme extract preparations (Schtzendbel et al., 2001). In industry, PVPP is widely marketed as a ning/stabilising agent, albeit a commercially expensive one, to remove polyphenols in stored beverage products (e.g. beer) in order to prevent protein-polyphenol haze formation and the consequently reduced shelf-life of the product (Dadic and Lavalle, 1983). In this study, we used Pb as the heavy metal and two sources of tannins for immobilisation onto PVPP: crude plant tannins from Symphytum ofcinale L. roots and commercially available puried tannins, in the form of tannic acid, from Chinese gallnuts. S. ofcinale was used because of the level of polyphenols in its roots, and also its high biomass, which is an attractive feature for phytoremediation (Lawerence, 1976).

2.3. Pb chelation to immobilised tannins 2.3.1. Overview of method In developing this method, Pb chelation experiments were conducted using tannins from S. ofcinale root tissues and puried tannins in the form of tannic acid (TA) from Chinese gallnuts. With respect to tannins from S. ofcinale root tissues two Pb chelation tests were conducted: one based on the in vivo differences in tannin levels between lateral and main root tissues (Section 2.3.2) and the other based on increasing concentrations of only one tissue type (Section 2.3.3). In all experiments PVPP was used specically to bind, immobilise, and therefore to separate tannins from root polyphenol extracts or puried TA from Chinese gallnut tannin solutions. Section 2.3.2 used a macro-scale version of the method in which 5 mL of tannin was immobilised with 0.1 g PVPP. Subsequent Section 2.3.3 and 2.4 used a micro-scale version of the method: 1 mL tannin was immobilised with 0.02 g PVPP. 2.3.2. Chelation of Pb to tannins from main and lateral roots of S. ofcinale Five tubes with 0.1 g of insoluble PVPP each were prepared. The following were then added to the respective tubes: 5 mL of 50% (v/ v) methanol (to Tube 1), 5 mL of lateral root polyphenol extract (to Tubes 2 and 3), and 5 mL of main root polyphenol extract (to Tubes 4 and 5). All ve tubes were then shaken horizontally for 20 min and then centrifuged at 10 000g at 10 C for 15 min. The liquid was removed for the tannin assay and the remaining PVPP pellet (now without or with immobilised root tannins) was rinsed twice with nanopure water. The following were then added to the PVPP pellets above. Five mL of 50 mg L1 Pb(NO3)2 solution (pH 4.5) were added to Tubes 1, 3, and 5. Tube 1 was the rst control to correct for any Pb chelation by PVPP; Tubes 3 and 5 were the Pb-tannin chelation tests. Five mL of 2% (v/v) HNO3 were added to Tubes 2 and 4 (the second control to correct for any background Pb in the PVPP-tannin pellet). All ve tubes were shaken for 4 h at room temperature under low light conditions and then centrifuged for 15 min at 10 000g at 10 C. The upper 2.5 mL layer of solution was digested with 30% (v/v) HNO3 in a 60 C water bath overnight (18 h). The level of Pb remaining in the upper layer of Tubes 3 and 5 was corrected with control Tubes 2 and 4. The level of Pb removed by immobilised tannins was calculated by comparing this gure with control Tube 1. This method was repeated for 250 and 500 mg L1 Pb(NO3)2. The concentration of Pb was determined using a ame AAS (model GBC Avanta R) with analytical grade Pb(NO3)2 as the standard. Note that the Pb treatment level refers to X mg L1 lead nitrate (Pb(NO3)2) where as Pb element or % Pb removed refers to X mg L1 Pb element as this was the form of Pb measured by AAS. 2.3.3. Chelation of Pb to increasing concentrations of lateral root tannins of S. ofcinale Only S. ofcinale polyphenols from lateral roots were used. The same protocol as Section 2.3.2 was applied, but in a micro-scale version and with the following modications: (i) extraction of polyphenols was at a ratio of 0.05 g root powder to 5 mL of 50% (v/v) methanol, (ii) tannins were immobilised using 0.02 g PVPP with 1 mL of polyphenol extract at increasing concentrations (0%, 12.5%, 25%, 50%, and 100% (v/v) of initial extract), (iii) one mL of 50 mg L1 Pb(NO3)2 (pH 4.5) was added to immobilised tannins and Pb chelation period reduced to 2 h at room temperature, and (iv) the level of Pb removed from solution was directly determined by AAS after diluting 0.5 mL of the top layer solution with 2 mL of 1% (v/v) HNO3.

2. Method and materials 2.1. Plant material Three-month-old S. ofcinale plants were grown from root cuttings in North-end sand supplemented with Nutricote 89-month slow-release fertiliser (Yates New Zealand Ltd., Auckland, New Zealand) at 5 g L1 of sand. All plants were grown under growth room conditions (16 h photoperiod at 26.5 lmol m2 s1, 22 C). Only root tissues were used in the experiments. Lateral root tissue is dened as the ne root tissue branching from the eshier main root tissue. 2.2. Polyphenol extraction and assay Freezedried, ball-mill ground lateral or main root tissues from 3-month-old S. ofcinale were used for all polyphenol extracts. Polyphenols were extracted at a ratio of 0.2 g of tissue to 15 mL of 50% (v/v) methanol with an ultrasonic water bath for 30 min. Samples were centrifuged at 10 000g for 30 min at 4 C and supernatants were used for polyphenol assays. Polyphenol and tannins were quantied using the FolinCiocalteau assay (FAO/IAEA Manual, 2002).

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2.4. Chelation of Pb to Chinese gallnuts tannins This section validates the new method outlined here, using puried tannins (TA) from Chinese gallnuts (Sigma) and the micro-scale version of the method in Section 2.3.2 to validate the new in vitro Pb-tannin chelation protocol described here. To match the Pb-binding capacity of PVPP-immobilised S. ofcinale root tannins, the Pb(NO3)2 concentration was set at 50 mg L1 and the concentration range of puried tannin set to between 0 and 10 mg mL1 TA. 2.5. Data analysis Pb data were expressed as the quantity of Pb lost, or remaining in solution, relative to the immobilised tannin concentration, as it was hypothesised that this factor governs the overall quantity of Pb chelated or removed. The actual tannin: Pb ratio was not calculated as it was considered that this might create misleading trends as the tannin concentration increases, the number of Pb binding sites could be reduced depending on the degree of binding to other compounds or polyphenol polymerisation. Experiments were replicated three times and statistics were performed using Statistix (Analytical Software, Tallahassee, FL). All data were analysed using one-way ANOVA and tested for homogeneity of variance (F-max) and comparison of means using the Tukey test. Values are means SE of three replicates and those not sharing the same letter are signicantly different (P 6 0.05). Graphical trendlines represent second-order polynomial ts. 3. Results 3.1. Level of polyphenols in S. ofcinale root tissues On a dry weight basis, lateral root tissue from 3-month-old S. ofcinale plants contained eight times more extractable polyphenols and 13 times more tannins than main root tissue (Fig. 1). Lateral roots contained on average 3.9% (dw) tannins. The different tannin concentration between the lateral and main root tissues were used to determine whether our in vitro chelation data were consistent with reported positive correlations between tannin level and metal accumulation in tissues.

3.2. Pb chelation to immobilised tannins 3.2.1. Chelation of Pb to tannins from main and lateral roots of S. ofcinale The mean level of tannins immobilised to PVPP in each tube (expressed as mg TA) were 2.62 mg TA and 0.19 mg TA for the lateral and main root polyphenol extracts, respectively. The immobilised lateral root extract with higher tannin levels removed more Pb than that of the lower-tannin main root extract. At 50, 250, and 500 mg L1 Pb(NO3)2 concentrations, an average of 1.1, 3.0, and 3.5 times more Pb was removed (from the upper layer Pb solution), respectively by chelation to immobilised lateral root tannins (Fig. 2). At 500 mg L1 Pb(NO3)2, immobilised lateral root tannins removed a mean of 74 mg L1 Pb (element) from the solution. The highest percentage of Pb chelated occurred at the 50 mg L1 Pb(NO3)2 concentration where 86% (on average) of the Pb (element) was removed by PVPP-immobilised lateral root tannins. 3.2.2. Chelation of Pb to increasing concentrations of lateral root tannins of S. ofcinale Using the micro-scale version of the method in Section 2.3.2, a range of concentrations of immobilised tannins was prepared from lateral root polyphenol extracts. This was to provide additional evidence of tannin involvement in the observed Pb chelation trends, rather than factors related to the type of root tissue (e.g. root mass and age). The same trend (as Section 3.2.1) in Pb chelation was observed; the higher the tannin concentration, the greater the level of Pb removal from solution (P 6 0.05). In this case, 0%, 6%, 10%, 24%, and 60% of the initial Pb (element) level in 50 mg L1 Pb(NO3)2 was removed by increasing concentrations of the lateral root immobilised tannins (Fig. 3a). 3.2.3. Pb chelation to tannins from Chinese gallnuts At 10 mg mL1 TA, where 7 mg TA was immobilised to the PVPP (Fig. 4), 70% of Pb (element) was removed from 50 mg L1 Pb(NO3)2 (P 6 0.05) (Fig. 3b). This demonstrates that PVPP-immobilised tan-

300 no extract lateral root

Polyphenol level (mg TAg-1 d.w. tissue)

70 60 50 40 30 20 10 0 lateral main
c d

Pb level inupper layer (mg L-1)

250 Polyphenol Tannin

main root

a ab

200
bc

150
cd

`
100
e d

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f I I h g

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250

500

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Fig. 1. Polyphenol and tannin levels extracted from 3-month-old Symphytum ofcinale root tissue. TA = tannic acid.

Pb(NO3)2 treatment level (mg L-1)

Fig. 2. Level of Pb remaining in the upper layer after treatment with PVPP, with or without immobilised tannins from 3-month-old Symphytum ofcinale lateral root or main root tissues, after 4 h. Solutions were all at pH 4.5.

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Fig. 3. Level of Pb remaining in the upper layer of 50 mg L1 Pb(NO3)2 (pH 4.5) after 2 h of treatment with increasing concentrations of PVPP-immobilised tannins: (a) 3month-old Symphytum ofcinale lateral root tannins and (b) puried Chinese gallnut tannins (in the form of TA). TA = tannic acid.

nins have the ability to directly chelate and remove Pb from solution. 4. Discussion 4.1. Pb chelation to immobilised tannins This research developed a new, reliable, and simple in vitro metal chelation method using PVPP-immobilised tannins. Moreover, tannin immobilisation to PVPP is not a chemical modication process such as aldehydic crosslinking between tannins and the binding matrix (Liao et al., 2004). It is an adsorption process and thus our PVPP-immobilised tannins may be easily related to the metal chelation properties of tannins in vivo. The degree of Pb chelation by immobilised tannins from puried TA from Chinese gallnuts and crude extracts from S. ofcinale roots were consistent with the level of tannins in the solution and/or tissues. With respect to immobilised tannins from Chinese gallnuts, a much higher concentration of this tannin was required to achieved a similar percentage of Pb chelation to S. ofcinale root-

derived tannins (7 mg compared with 0.23 mg TA per tube). The increase may be in part due to the different plant origins of the tannin, use of puried versus crude mix of tannins and/or because S. ofcinale root tannins are oxidised or polymerised forms of tannins, which therefore under-estimates the effective tannin concentration. None the less, together these results corroborate recent quantitative studies suggesting the role of polyphenols in tolerance and accumulation to heavy metals in non-hyperaccumulating plants (Lavid et al., 2001; Kamachi et al., 2005). Between 0.5 and 1 mg kg1 Pb (dw) is sufcient to cause toxicity to plants. In this study, Pb bound by S. ofcinale tannins made up about 2000 mg kg1 (dw) of S. ofcinale lateral root tissue. The presence of more Pb in these tissues suggest that there is a biologically meaningful level of protective or tolerance-based chelation in S. ofcinale. The function of tannins is also likely to be important considering that they possess abundant metal-chelating hydroxyl groups and are the fourth most abundant metabolite in vascular plants after cellulose, hemicellulose and lignin (Kraus et al., 2003). Moreover, tannins are precursors of lignin, a known Pbbinding compound (Marmiroli et al., 2005). Determining their role, with respect to other known potential Pb-binding compounds such as phosphates, pectin, and lignin, in Pb accumulation and tolerance would be an interesting future study. 4.2. Role of Pb-tannin chelation in phytoremediation In any type of phytoremediation, the presence of free heavy metals is inevitable and potentially toxic to plant cells. Thus regardless of the type of phytoremediation, chelation of Pb by tannins would provide an important Pb tolerance and accumulation mechanism. Compared to the synthesis of specic bio-molecules for chelation (e.g. phytochelatins), it may also be a more energy efcient form of tolerance as it uses more permanent and premade bio-molecules for chelation (Marmiroli et al., 2005). Chelate-assisted phytoextraction, rhizoltration and phytostabilisation types of phytoremediation stand to benet most from Pb-tannin chelation. For example in chelate-assisted phytoextraction, EDTA has been recently added in increments rather than in a single dosage to enhance shoot accumulation of metals and to reduce PbEDTA leaching into the soil (Barocsi et al., 2003). Chelation of Pb by shoot tannins could help plants tolerate increasing amounts of PbEDTA in the shoot over a longer phytoextraction period. This could be especially important considering that up to 40% of the PbEDTA complex formed can dissociate (Geebelen et al.,

8 7

y = -0.06x + 1.32x - 0.41 2 R = 0.9994

b

mg TA bound to pellet

6
c

5 4 3 2 1 0 0 2 4 6 8 10 12
e d

Tannic acid level (mg mL-1)

Fig. 4. Level of puried Chinese gallnut tannins (in the form of TA) bound to 0.02 g of PVPP pellet. TA = tannic acid.

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2002), producing free toxic Pb. Phytostabilisation and rhizoltration (where root-based metal accumulation is targeted) can be enhanced by specically selecting plants with roots that are high in biomass and tannin content. Tannins could, respectively contribute to the Pb-binding matrix in the soil after roots die or act as a matrix for binding the Pb in roots that are in contact with heavy metal solutions. 5. Conclusions Using tannins extracted from S. ofcinale roots and puried tannins (TA) from Chinese gallnuts, a new, reliable and simple in vitro Pb chelation protocol was developed that directly demonstrated the ability of tannins to chelate Pb ions. This method showed that the levels of tannins in S. ofcinale roots, and in the immobilised matrix, were in each case reected in the quantity of chelated Pb. The simplicity of this method should make it widely accessible and applicable to other metal ions and to tannins from other plant species. The provision of evidence supporting the role of Pb-tannin chelation in Pb tolerance and accumulation is an important step in the development of successful phytoremediation protocols. The selection of plants for screening for a particular type of phytoremediation should therefore take into account the presence, levels and tissue distribution of metal-chelating phytochemicals such tannins in addition to other factors such as biomass. Acknowledgements The authors would like to acknowledge the support of the Charles Alma Baker Trust for the support of a doctoral scholarship and helpful comments from the reviewers. References
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